RU2430969C2 - High-polymer rna from baker's yeast, free from impurity associations of rna with proteins and polysaccharides - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: disclosed is a preparation of high-polymer RNA from baker's yeast. The high-polymer RNA is extracted using sodium dodecyl sulphate. RNA is salted out with sodium chloride. Washing is carried out at low centrifuging rate of 1000 - 3000 g for 10 minutes without cooling.

EFFECT: obtained preparation is free from impurity association of RNA with proteins and polysaccharide and has narrow-directed stimulation of hemapoiesis.

1 dwg

Description

The invention relates to medicine, in particular to the use for therapeutic purposes of new physiologically active compounds.

Known drug is a high polymer RNA extracted from baker's yeast cells using sodium dodecyl sulfate (DDS-Na) [1]. It has been produced according to this technique since 1955 in preparative quantities.

However, as can be seen from drawing (a), published for the first time in [2], this high-polymer yeast RNA preparation contains a significant amount of impurity RNA associates with proteins and polysaccharides. In the presence of this impurity, high-polymer yeast RNA acts on the organs and tissues of laboratory animals very non-specifically and practically does not stimulate hemoglobin biosynthesis [3].

The aim of the present invention is to obtain a highly polymeric yeast RNA capable of narrowly stimulating hematopoiesis.

This goal is achieved by removing impurity RNA associates from proteins and polysaccharides from the RNA isolated by low-speed centrifugation at the stage of RNA salting out with sodium chloride and subsequent washing. Studies have shown that with centrifugal acceleration from 1000 to 3000 g, large flakes of salted high-polymeric RNA precipitate to form a cake - meal, and the small haze of RNA associates with proteins and polysaccharides remains in suspension and is removed by decantation; at acceleration more than 3000 g they co-precipitate; when accelerating less than 1000 g, the sediment - meal forms loose and reaches for the decanted supernatant. The optimum - 1500 g (10 min, without cooling) was implemented in the proposed method.

Characteristics of the obtained preparation: yield from 30 g of dry yeast - 0.7-1.0 g; the color of the lyophilized preparation is white; solubility in water is good; weight extinction, OE ₂₆₀ / mg - 19-21; CRF content,% ≤2.5; an increase in CRF for 1 day at 22-25 ° C in distilled water,% ≤0.5; spectral ratios: D ₂₃₀ / D ₂₆₀ - 0.35-0.55; D ₂₅₀ / D ₂₆₀ - 0.86-0.94; D ₂₈₀ / D ₂₆₀ - 0.4-0.55.

An example implementation of the proposed method

30 g of fresh dry baker's yeast (Saccharomyces cerevisiae, Novosibirsk Yeast Plant, GOST 28483-90) with a moisture content of ~ 7.4% was suspended in portions for 1-2 minutes in 0.3 l of boiling water containing 7.5 g of DDS -Na (Scientific-Production Company "DIA-PHARM", Belgorod), so that the temperature of the suspension does not fall below 99 ° C. The suspension was boiled for 40 minutes at 99-102 ° C. Periodically stirring. As evaporated, boiling water up to 0.3 l was added to it. At the end of the extraction, the suspension was cooled to ~ 40 ° C, centrifuged (1500 g, 10 min, without cooling), the supernatant was poured into a 0.5 L glass container, 53 g of NaCl was added to it (FSUE of SIBSOL plant, Usolye -Siberian, Irkutsk region, GOST R 51574-2000) and fresh distilled water up to 0.3 l. The suspension was vigorously stirred until the salt was completely dissolved and left at room temperature for 22 hours to form a precipitate. The sediment salted with large flakes, meal, containing the target product - high polymer RNA - was separated from the supernatant containing impurity RNA associates with proteins and polysaccharides in the form of fine turbidity by low-speed centrifugation (1500 g, 10 min, without cooling), washed with 2 portions of 0 , 2 l of 3 M NaCl solution and 2 portions (0.1 and 0.05 l) of 94% ethanol (Alcohol Plant OJSC, Mariinsk, Kemerovo Region, GOST R 51652-2000); while the meal was separated from a 3 M solution of NaCl and ethanol by low-speed centrifugation (1500 g, 10 min, without cooling).

Commodity high-polymer RNA, free of impurity RNA associates with proteins and polysaccharides, was obtained by extracting it from washed meal with distilled water, clarifying the extract by filtration through a POR-100 glass porous filter and final lyophilization.

As can be seen from drawing (b), the obtained preparation of high-polymer yeast RNA (the alleged market name "Vitalang-1") is free from impurity RNA associates with proteins and polysaccharides. When administered intraperitoneally to laboratory animals, it specifically affects exclusively red bone marrow cells, which strongly stimulate hematopoiesis [3].

Information sources

1. Crestfield A.M., Smith K.C., Alien F.W. The preparation and characterization of ribonucleic acids from yest // J. Biol. Chem., 1955, V.216, N1, P.185-193.

2. Yamkova T.V., Khomov V.V., Zagrebelny S.N., Yamkova V.I. Isolation of total RNA from baker's yeast // Prikl. Biochemistry and Microbiology, 2006, T.42, N1, S.93-97.

3. Yamkova T.V., Yamkova V.I., Panin L.E. The biological activity of different preparations of RNA from baker's yeast // Bull. Siberian Branch of the Russian Academy of Medical Sciences, 2006, No. 3 (121), pp. 117-121.

Claims (1)

High-polymer RNA preparation isolated from DDS-Na from baker's yeast, characterized in that it is freed from impurity RNA associations with proteins and polysaccharides by salting out RNA with sodium chloride and subsequent washing with low-speed centrifugation at 1000-3000 g for 10 min without cooling.

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